Extracellular-Matrix Mechanics Regulate the Ocular Physiological and Pathological Activities

The extracellular matrix (ECM) is a noncellular structure that plays an indispensable role in a series of cell life activities. Accumulating studies have demonstrated that ECM stiffness, a type of mechanical forces, exerts a pivotal influence on regulating organogenesis, tissue homeostasis, and the occurrence and development of miscellaneous diseases. Nevertheless, the role of ECM stiffness in ophthalmology is rarely discussed. In this review, we focus on describing the important role of ECM stiffness and its composition in multiple ocular structures (including cornea, retina, optic nerve, trabecular reticulum, and vitreous) from a new perspective. The abnormal changes in ECM can trigger physiological and pathological activities of the eye, suggesting that compared with different biochemical factors, the transmission and transduction of force signals triggered by mechanical cues such as ECM stiffness are also universal in different ocular cells. We expect that targeting ECM as a therapeutic approach or designing advanced ECM-based technologies will have a broader application prospect in ophthalmology.


Introduction
A large number of studies are increasingly concerned with the physical properties of cells and tissues along with the infuences of biophysical characteristics within tissue microenvironments on cell function [1][2][3]. Tere is growing evidence that external mechanical factors impact the confguration of biomacromolecules, the fate of cells, and the structure of tissues [4,5]. Te extracellular matrix (ECM) is a common scafold for maintaining the homeostasis of tissues and organs [6]. It forms a complex but highly organized network and is remodeled dynamically around cells that not only provides mechanical support for the completeness and resilience of cells but also modulates cell homeostasis and signal transduction [7][8][9]. ECM is a crucial component of the ocular microenvironment, which plays an essential role in every part of the eye, either maintaining the transparency and hydration of the cornea and vitreous, or modulating angiogenesis, intraocular pressure (IOP) maintenance, and vascular signaling [10,11]. ECM is the noncellular structure composed of water, collagens, and glycoproteins, and all tissues have the ECM with an individual composition and topology, which is developed through the crosstalk and interplays of biochemical and biophysical between manifold cellular components (such as epithelial, fbroblast, adipocyte, endothelial elements) as well as a constantly remodeled cellular and protein microenvironment during tissue development [12][13][14].
ECM stifness, a source of mechanical stimulation, transfers the external physical force onto the cell, which is transformed into biochemical signals through a series of cascade reactions inside the cells to regulate various ocular physiological and pathological activities, including cell migration and proliferation, retinal vascular development, corneal homeostasis, maintenance of normal human trabecular mesh function, and IOP as well as the occurrence of glaucoma and subconjunctival fbrosis [12,[15][16][17].
In this review, we summarize the major components and physical properties of ECM and its underlying mechanisms for regulating normal ocular physiological and pathological activities. We aim to provide new insights into how the multiple signals are integrated to modulate ocular cell behaviors for the development of novel therapeutic strategies for ocular tissue repair and regeneration while targeting ECM as a therapeutic modality.

ECM Stiffness in the Cornea
Te cornea is a transparent tissue located on the anterior surface of the eyeball and is the eye's major refractive structure, providing most of the refractive power needed to focus light onto the retina [18]. Te cornea is composed of the outer epithelium, the stroma, and the inner endothelium, of which the stroma accounts for more than 90% of the corneal thickness [19]. Te corneal stroma is an ECM with enrichment of collagen and highly ordered characteristics, which provides clarity and preserves the structure necessary for light refraction after assembly [18,20]. In addition, the abundance of collagen I in the ECM confers the cornea in biomechanical stability and form [21,22]. Corneal keratocytes are stroma-resident cells in charge of maintaining the ECM's highly organized structure and the homeostasis of its constituent parts [21,23]. After surgery or trauma, the mechanical characteristics of the cornea will change signifcantly during wound healing, leading to a substantial increase in ECM stifness [24][25][26]. Tis is due to the fbrotic reaction caused by the activation of corneal cells from a natural mechanically quiescent condition to the active myofbroblast state, which distorts the highly ordered structure of the ECM, resulting in corneal turbidity and even visual impairment [15]. Since it has been shown that transforming growth factor-β1 (TGF-β1) is released into the stromal space after injury and can promote the diferentiation of dormant keratocytes into myofbroblasts, the phenotypic transition of corneal keratocytes is linked to signaling downstream of TGF-β1 [27][28][29]. Primary corneal keratocytes were cultured on collagen-coated glass coverslips or a stif (10 kPa) gel matrix, which resulted in mechanical dependence with a wider variety of morphology, plentiful stress fber formation, higher levels of α-smooth muscle actin (α-SMA) expression, and stronger traction forces [15,30]. Conversely, corneal cells cultivated on a soft (1 kPa) gel produced fewer stress fbers and kept more of their dendritic morphology, indicating a dormant keratocyte phenotype [15]. Tese results emphasize the crucial role that ECM stifness plays in controlling the mechanical phenotype of corneal cells during corneal wound healing.
Te biomechanical properties of the cornea, including ECM stifness, have recently been used in clinical risk assessments to diagnose glaucoma and predict disease progression [31,32]. Corneal biomechanics change with age and loss of stromal tissue was observed in corneal pathology [33,34]. Te three key proteins lysyl oxidase (LOX), transglutaminase-2 (TGM-2), and advanced glycation end products (AGEs) are responsible for more collagen crosslinking, which causes an excessive increase in ECM stifness and causes ocular stifness in glaucoma, refecting the signifcant role ECM plays in the mechanical homeostasis of the eye [31,35,36].

ECM Stiffness in the Retina
Mammalian vision begins with the transmission of light through the cornea and lens to the retina, which is the layer of cells along the rear wall inside the eye [37]. Te extremely professional retina fnishes the conversion of energy from absorbed photons into neural activities, and the brain consequently can elucidate the patterns of detected photons [37,38]. ECM molecules are found mostly in the basement membrane (BM) and nonbasement membrane (NBM) of the retina [39]. BM is linked to three types of retinal tissue: Bruch's membrane, inner limiting membrane (ILM), and vasculature, and their major physiological role is to demarcate the neural retina from surrounding non-neural tissue [16,40,41]. Typically, the NBM is usually located between the retinal pigment epithelium (RPE) and the ILM [16].
In the retina, ECMs are widely distributed throughout the nerve fber layer, the outer and inner plexiform layers, and the interphotoreceptor matrix [41]. Muller glial cells, intraretinal glial cells as well as migrating astrocytes represent the major sources of ECM secretion [16,42,43]. ECM creates the environment around retinal cells, serves as the BMs, and ofers structural and mechanical support. Meanwhile, its constituents are essential for the diferentiation and development of the retina [16,44]. Te development of retinal microvessels is regulated by the laminin family, and alterations in the collagen, elastin, and tenascin-C content of the ECM will increase axonal injury [45]. Following ischemia, neurodegeneration of the retina and optic nerve is associated with the remodeling of several ECM molecules [44]. Te elastic modulus of retinal cells ranges from 200 Pa to 1000 Pa, and a variety of pathological reactions in the retina lead to local variations in retinal tissue stifness [46]. Te considerable changes in retinal rigidity brought on by age, laser surgery, and retinal detachment are mostly attributed to the ECM [47]. Excess ECM deposition leads to increased ECM stifness, inducing the activation of RPE cells together with the associated complement system, which in turn promotes the epithelial-mesenchymal transition (EMT) of RPE cells to initiate subretinal fbrosis (Figures 1(a) and 1(b)) [48,49]. Strikingly, the underlying mechanism is that ECM stifness regulates RhoA GTPase activity and the actin cytoskeleton to activate mechanically sensitive molecules Yes-associated protein/transcriptional coactivator with pdz-binding motif (YAP/TAZ) and then drives their translocation into the nucleus, where they interact with the TEAD family of transcription factors to form the complex (Figure 1(c)) [47]. Intracellular distribution and expression of YAP are regulated by ECM-related mechanical stifness, which is associated with pathological fbrosis, and overexpression of YAP has been detected in renal, lung, and liver fbrosis [47]. Te transcription complex then regulates RPE cell migration, proliferation, and contraction through target genes that are implicated with the development of proliferative vitreoretinopathy (PVR) (Figure 1(c)) [47].
In vitro experiments found that compared to cells cultivated on the soft gel (0.5 kPa) matrix, cultured ARPE-19 cells (human retinal pigment epithelial cell line) on rigid (50 kPa) gel coverslips showed signifcantly increased ECM production and nuclear translocation of YAP as well as signifcantly elevated protein levels of its downstream targets, connective tissue growth factor (CTGF), and CYR61, which are the signaling molecules associated with retinal fbrosis (Figure 1(c)) [47,50]. In addition, it was shown that inhibiting YAP and RhoA reduced the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 induced by ECM stifness in the retina. Tese results suggest that targeting ECM or blocking YAP and RhoA signaling pathways can reduce PVR-induced retinal fbrosis and

ECM Stiffness in the Trabecular Meshwork
Trabecular meshwork (TM) provides the majority of the fow resistance to the outfow of aqueous humor, and the manipulation of trabecular outfow resistance is responsible for the modulation of IOP [51,52]. ECM is an important constituent of all parts of the TM: the corneoscleral, uveoscleral, and juxtacanalicular layers [10,53]. ECMs in TM consist of glycosaminoglycans and proteoglycans, collagens, elastic fbrils, basement membrane, and matrix proteins, and they determine the TM stifness [51,54]. Te outfow channel of ECM is quite dynamic and undergoes continuous turnover and remodeling, which is contributing to regulating the homeostasis of IOP (Figures 2(a) and 2(b)) [55,56]. Environmental stimulation, such as mechanical stretch or a series of growth factors, cytokines, and drugs (such as dexamethasone), will alter the expression of ECM components and its mechanical properties, triggering the continuous increase of trabecular outfow resistance, leading to the enhanced IOP, which is the main risk factor for glaucomatous optic neuropathy and a link to deteriorating vision [51,57,58]. Te progression of primary open-angle glaucoma (POAG) was associated with the increased stifness of human TM (HTM) and enhanced levels of transforming growth factor-β2 (TGF-β2) in aqueous humor [59]. ECM stifness has been shown to profoundly alter the cytoskeletal structure and kinetics of HTM [60,61]. Trough the biomimetic ECM hydrogels or polyacrylamide substrates with tunable stifness to explore the mechanism of ECM regulation of glaucoma, substrate stifness signifcantly enhanced TM cell spread and altered TM cell morphology [59,62]. Multi-omics analysis and immunostaining revealed that prominent stress fbers observed on stif substrates were related to the formation of focal adhesion and that a distinct stifness-induced reorganization of the actin cytoskeleton [59,63]. Stifer substrates also promoted increased TM cell proliferation. Moreover, increased substrate stifness was found to alter the intrinsic TM cell stifness profle (Figures 2(a)-2(c)) [64]. More importantly, stifer ECM hydrogels up-regulated TGF-β2 expression and YAP/TAZ activity as well as their translocation in the nucleus [59]. TGF-β2 has been shown to induce nuclear YAP/TAZ localization and the consequent activation of target genes (which are correlated with the development of glaucoma), including TGM-2, CTGF, and plasminogen activator inhibitor-1 (PAI-1), via ERK and ROCK signaling pathways in response to mechanical signals (Figure 2(c)) [59,65,66]. Of note, the Wnt signaling avenue, another vital mechanism in glaucoma, is involved in regulating ECM stifness in response to mechanical signals infuenced by YAP/TAZ (Figure 2(c)) [67]. Te antagonistic efects of Wnt signaling are a cause of elevated IOP, or the expression of component Wnt is crucial for preserving normal IOP in HTM (Figure 2(c)) [66]. Tese results confrm the impact of ECM stifness on TM cells, and elevated stifness may contribute to the fbrotic behavior of TM cells in glaucoma, suggesting that therapies targeting ECM or ECM-related mechanical signaling molecules can be developed to prevent the occurrence and development of glaucoma.

ECM Stiffness in the Optical Nerve
ECM is constantly remodeled in the optic nerve, and its various components, including glycoproteins fbronectin, laminin, tenascin-C, and tenascin-R as well as the chondroitin sulfate proteoglycans (CSPGs), aggrecan, neurocan, and brevican, are critical for maintaining the normal function of the optic nerve [68][69][70]. Tenascin-R can modulate neurite outgrowth as well as neural and glial adhesion, whereas tenascin-C is increased and engaged in neuroinfammation and glial response under pathological conditions, and CSPG is highly aggregated in glial scars, limiting axon's ability to regenerate [44,71]. Axon damage can be exacerbated by changes in ECM composition, and remodeling of several ECM components is linked to neurodegeneration of the retina and optic nerve following ischemia [16,71]. Te retinal ganglion cell (RGC) axons' egress as well as the entrance and exit of the retinal blood vessels are made possible by the optic nerve head (ONH), a structure in the posterior ocular fundus. Collagenous loadbearing beams make up the lamina cribrosa (LC), a porous support system that spans the ONH and provides protection for fragile, unmyelinated RGC axons as they depart the eye posteriorly to form the optic nerve [72,73]. LC is made up of ONH astrocytes and fbroblast-like cells (referred to as LC cells), which are essential for maintaining the surrounding ECM [72,74].
Signifcantly larger cell spread area and enhanced actin flament growth and the creation of more vinculin-focal adhesions (number and size) were observed in both normal and glaucoma LC cells cultured on stif silicon elastomer surfaces (100 kPa) [74]. Tese alterations were shown to be positively correlated with enhanced cell stifness as evaluated by atomic force microscopy (AFM) [75,76]. Proliferation and cytoskeletal alterations in glaucoma LC cells were noticeably greater than in normal cells. Both LC cells exposed to a stifer substrate underwent the change into a myofbroblast-like phenotype, as shown by enhanced α-SMA signaling and its colocalization with actin stress fbers [74]. Interestingly, normal LC cells cultured on a mimicked stifness substrate (100 kPa) showed signifcantly upregulated YAP gene and protein expression, followed by elevated YAP phosphorylation at tyrosine 357, and decreased YAP phosphorylation at serine 127 ( Figure 3). Tis diferential phosphorylation expression results in an enhancement in total-YAP with an increase in nuclear translocation and aggregation as well as a decrease in nuclear export, thus promoting the transcriptional activity of YAP/ TEAD complex transcription targets, which leads to elevated ECM protein synthesis, enhanced myofbroblast markers, augmented activation of connective tissue growth factors, and increased proliferation, eventually forming a feedforward cycle mode (Figure 3) [72]. Notably, treatment with the YAP-specifc inhibitor verteporfn remarkably disrupted Journal of Ophthalmology mechanotransduction in LC cells triggered by enhanced ECM stifness, eliminating any subsequent pro-fbrosis processes and positive feedback loops [72].
Tese results showed that a stifer cell microenvironment stimulates a myofbroblastic transition in human LC cells, thus contributing to LC remodeling and fbrosis in glaucoma.

The Role of ECM in the Vitreous Body
Te human vitreous body is a nearly spherical transparent structure, about 4.5 milliliters in volume, which is encircled by and attached to the retina, pars plana, and lens of the eye [77]. Te vitreous body is a highly hydrated, almost acellular, transparent tissue with various ECM components, including thin heterotypic collagen fbers composed of type II, type IX, and type V/XI collagen that are crucial to the gel structure [78,79]. Hyaluronan is the main glycosaminoglycan in the mammalian vitreous body, which forms a reticular structure to support collagen fber scafold and allows the expansion of gel through swelling osmotic gradient [10,78]. Te human vitreous body is gel-like at birth, but with the growth of age, it will experience an inevitable liquefaction process, and collagen fbers aggregate during age-related vitreous liquefaction [80,81]. Changes in the composition of ECM and fbrous aggregation appear to be the main events of agerelated vitreous liquefaction, thus may result in the weakening of vitreoretinal adhesion and the posterior vitreous detachment, which is implicated in the pathogenesis of many blinding ocular diseases, including rhegmatogenous retinal detachment [77,78].

ECM-Based Technology to Treat Ocular Diseases
By 2020, hundreds of millions of people around the world have diferent degrees of visual impairment as a result of eye diseases and trauma [82]. It is urgent to develop advanced medical strategies to promote benefcial tissue remodeling in the nerve tissue of the eye, retina, and optic nerve. Te biological scafold composed of ECM derived from the decellularized processes of diferent mammalian tissues or organs has retained many bioactive molecules unique to natural tissues, including collagen, glycosaminoglycans, When ECM stifness increases with aging or glaucoma, F-actin increases followed by YAP phosphorylation at tyrosine 357 elevates, while its serine 127 phosphorylation decreases, but the total YAP augments, resulting in elevated nuclear translocation and enhanced nuclear import (to some extent through direct F-actin-mediated pore opening) as well as lowered nuclear export, which correspondingly promotes the transcriptional activity of targets, leading to upregulation of ECM protein expression, enhanced myofbroblastic markers, incremental activation growth factors, and increased proliferation [72]. A "positive feedback loop" on the right illustrates how this cycle is self-sustaining. CTGF: connective tissue growth factor. α-SMA: α-smooth muscle actin. COLA1: α-1 type I collagen. c-myc: the regulator genes and proto-oncogenes that code for transcription factors.
laminin, and growth factors [83][84][85][86] (Figure 4(a)). It has been successfully used in clinical practice to promote the constructive remodeling of many tissues, including skin, heart, esophagus, bladder, and muscle [87,88]. At present, researchers have gradually applied ECM technology to the feld of ophthalmology and developed ECM hydrogel technology and ECM biological hybrid scafold [87,89].

ECM Hydrogels.
A retrobulbar, periocular, or even intraocular injection can be used to distribute ECM-derived hydrogels since they are organic, biocompatible medical devices with the potential for minimally invasive administration (Figures 4(a) and 4(b)) [87,90]. In the case of penetrating ocular injuries, local ECM hydrogel injections provide a biocompatible, adjustable gel for bridging acellular  injury-induced gaps, enlisting and guiding endogenous stem cell localization and development, and regulate the immune system to accelerate active tissue rebuilding [87,91]. In the meanwhile, a potential treatment for maintaining visual function is the transplantation of purifed retinal progenitor cells or neural stem cells (NSCs) based on the ECM hydrogel platform [87,92,93]. Injecting RGCs, progenitor cells, or stem cells intravitreally or into the optic nerve is minimally invasive and has demonstrated neuroprotective benefts in various models of neurodegenerative illness (Figures 4(a) and 4(b)) [94,95]. Transplanted RGC or stem cells are supposed to retard the onset of retinal degeneration by concurrently controlling a number of pro-survival mechanisms via locally produced neurotrophic factors and/or intraocular microenvironment regulation (Figures 4(a) and 4(b)) [96,97]. Interestingly, the ocular delivery of collagen mimetic peptide (CMP) compensates for the damage caused by the biochemical degradation and remodeling of ocular ECMs, preventing the functional defect of RGC projected to the brain after the increase of IOP in the most prevalent model of experimental glaucoma, microbead occlusion (Figures 4(a) and 4(b)) [98,99]. Tis suggests that intravitreal injection of CMP incorporating the tissue-specifc advantages of the ECM hydrogel platform may afect the extensive repair of ECM and promote RGC survival, providing an alternative for patients with chronic and acute optic neuropathy [87,98].

ECM Biological Hybrid Scafold.
Combining the tunable mechanical and biochemical properties of an electrospun polymer sheet with ECM or ECM-derived meterials, endowing the bio-hybrid scafold the advantages of higher tensile strength and controllable degradation rates [87,100]. Researchers have used electrospinning to generate a scafold with aligned fbers that directionally guide the growth of retinal ganglion axons, which is benefcial for the treatment of glaucoma and other optic neuropathies through cell transplant therapies [101]. Biohybrid scafolds, which incorporate ECM hydrogels, have stronger biocompatibility than solely synthetic scafolds and may ofer tissue-specifc benefts for improving neuropathies and contributing to functional recovery after retinal diseases [87].

Conclusion
ECM provides attachment and adhesion for cells, regulates intercellular communication, and supports the integrity of multicellular structures. ECM is highly tissue-specifc, and its components vary with multicellular structure, afecting a variety of cell life processes. ECM stifness is a form of mechanical force, and mechanical mechanics is an essential factor in the modulation of cell function and behavior. However, the role of ECM stifness is rarely discussed in the feld of ophthalmology. In this paper, we focus on describing the important role of ECM stifness and its composition in multiple ocular structures (including cornea, retina, optic nerve, trabecular meshwork, and vitreous body) from a new perspective. Abnormal changes in ECM stifness and its composition can trigger physiological and pathological activities of the eye in response. Tis also suggests that, compared with diferent biochemical factors, ECM stifness, a mechanical cue, is universal in the transmission and transduction of force signals in diferent ocular cell types. In addition, we summarized the advantages of using ECM technology as regenerative medicine in the treatment of ocular diseases and emphasized that ECM has great research value and clinical application prospects in the feld of ophthalmology.

Data Availability
Te data analyzed in this study is included in this manuscript, and the references supporting the fndings of this study are included witin this article.

Conflicts of Interest
All authors declare that there are no conficts of interest.